Recent investigations have identified a metabolic pathway involving sphingomyelin and derivatives that may be involved in signal transduction [1-8]. This pathway is initiated by the hydrolysis of sphingomyelin to ceramide via the action of a sphingomyelinase. Ceramide may then be deacylated to sphingoid bases, putative inhibitors of protein kinase C [9-12], or phosphorylated to the sphingolipid ceramide 1-phosphate by the action of a recently described calcium-dependent ceramide kinase [4, 5, 13]. The biologic role of ceramide 1-phosphate and regulation of the kinase that mediates its synthesis have not yet been determined. This pathway appears specific for ceramide derived from sphingomyelin, as ceramide derived from glycosphingolipids is not converted either to sphingoid bases [14] or to ceramide 1-phosphate [4].
Recently, Hannun and coworkers [6-8] have provided evidence that this sphingomyelin pathway may be involved in signal transduction. Tumor necrosis factor (TNF) .alpha., .gamma. interferon, and 1,25-dihydroxyvitamin D.sub.3, factors that induce monocytic differentiation of HL-60 promyelocytic cells, all stimulate sphingomyelin degradation to ceramide as an early event in cellular activation [6-8]. A synthetic ceramide N-acetylsphingosine could replace these agents in induction of monocytic differentiation of these cells. Furthermore, there have also been numerous reports that TNF and IL-1 stimulate a common set of events in diverse biological systems [60].
Direct evidence for second-messenger function for ceramide has also been shown. Davis and coworkers [15-17] originally showed that sphingosine induced epidermal growth factor receptor (EGFR) phosphorylation on Thr-669 in A-431 human epidermoid carcinoma cells by a mechanism that did not involve protein kinase C. It was demonstrated that sphingosine was rapidly converted to ceramide by these cells and that ceramide induced identical phosphorylation [18]. These studies were interpreted as evidence that ceramide had bioeffector properties, and might mediate, in part, the action of exogenous sphingosine. However, prior to the subject invention, no kinase was identified capable of mediating the effects of ceramide as a second messenger.
The subject invention provides a purified ceramide-activated protein kinase which functions as a key element in a sphingomyelin pathway utilizing ceramide as a second messenger. The knowledge that a ceramide-activated protein kinase exists as part of the sphingomyelin pathway enables the treatment of certain disorders by selectively modifying the function of this kinase in appropriate cells. Such disorders where this approach is possible include, by way of example, HIV infection, inflammatory disorders and disorders associated with poor stem cell growth. Accordingly, the subject invention provides methods of treating subjects having such disorders with agents capable of modifying the activity of ceramide-activated protein kinase, and methods of identifying such agents.